Development of Nonaggregating Poly-A Tailed Immunostimulatory A/D Type CpG Oligodeoxynucleotides Applicable for Clinical Use.

Taiki Aoshi,Yuko Katakai,Etsushi Kuroda,Hideaki Sato,Cevayir Coban,Kazuo Sakurai,Kouji Kobiyama,Hirokazu Nankai,Hirotaka Narita,Shinichi Mochizuki,Yasuhiro Yasutomi,Ken J Ishii,Yasunari Haseda

doi:10.1155/2015/316364

Abstract

Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

Highlights

Immunostimulatory CpG oligodeoxynucleotides (ODNs) have been developed and utilized as Toll-like receptor (TLR) 9-dependent innate immune activators and vaccine adjuvants for more than 10 years [1]
Cytokine enzyme-linked immunosorbent assay (ELISA) revealed that D35 (A/D type CpG-ODN) with an additional dAs40 tail at the 3󸀠 end was active as the original D35 (Figure 1(a); sample 1 versus D35)
Subsequent experiments further revealed that some D35-derived ODNs with a dAs40 tail were biologically active, even with replacement of the 3󸀠 Ghexamer to A, T, and C-hexamer (Figure 1(a); samples 5, 9, and 13)

Summary

Introduction

Immunostimulatory CpG oligodeoxynucleotides (ODNs) have been developed and utilized as Toll-like receptor (TLR) 9-dependent innate immune activators and vaccine adjuvants for more than 10 years [1]. Based on their backbone and sequence characteristics, immunostimulatory ODNs can be divided into four proposed different types (or classes) [2, 3]: A/D, B/K, C, and P-type ODNs. A/D types ODNs (mostly phosphodiester backbone with poly G tail at 3󸀠 end) mainly stimulate interferon- (IFN-) α production from plasmacytoid dendritic cells (pDCs). P-type ODN (all phosphorothioate backbone) contains two palindromic sequences with a cytokine profile similar to C type ODNs but with higher IFN-α production [4]

Methods

Results

Conclusion